Identification of the gene causing the mouse scurfy phenotype and its human ortholog

ABSTRACT

Isolated nucleic acid molecules are provided which encode Fkh sf , as well as mutant forms thereof. Also provided are expression vectors suitable for expressing such nucleic acid molecules, and host cells containing such expression vectors. Utilizing assays based upon the nucleic acid sequences disclosed herein (as well as mutant forms thereof), numerous molecules may be identified which modulate the immune system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.10/115,195, filed Apr. 2, 2002, now U.S. Pat. No. 6,884,618, which is aContinuation of U.S. patent application Ser. No. 09/372,668, filed Aug.11, 1999, now U.S. Pat. No. 6,414,129, which claims priority from U.S.Provisional Application No. 60/096,195, filed Aug. 11, 1998, whichapplication is incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to pharmaceutical products andmethods and, more specifically, to methods and compositions useful fordiagnosing scurfy-related diseases, as well as methods for identifyingcompounds which can modulate the immune system.

BACKGROUND OF THE INVENTION

Inherited mutations affecting the murine immune system have proven to bea rich source of novel genes critical to the regulation of the immunesystem and have furnished important animal models for humanimmunological disorders. These include xid, the murine equivalent ofX-linked agammaglobulinemia (Thomas et al., Science 261:355, 1993;Rawlings et al., Science 261:358, 1993), beige (the equivalent ofChediak-Higashi Syndrome) (Barbosa, et al., Nature 382:262, 1996), lprand gld (defects in fas and fas-ligand), X-linked severe combinedimmunodeficiency (Sugamura et al., Annu. Rev. Immunol. 14:179, 1996),and the hematopoietic cell phosphatase mutant motheaten (SHP-1) (Bignonand Siminovitch, Clin Immunol Immunopathol 73:168, 1994).

One mouse mutant of particular interest is the as-yet uncloned X-linkedmouse mutant, scurfy (sf). Briefly, mice hemizygous for the scurfymutation exhibit a severe lymphoproliferative disorder. In particular,males hemizygous (X^(sf)/Y) for the scurfy mutation develop aprogressive lymphocytic infiltration of the lymph nodes, spleen, liverand skin resulting in gross morphological symptoms which includesplenomegaly, hepatomegaly, greatly enlarged lymph nodes, runting,exfoliative dermatitis, and thickened malformed ears (Godfrey et al.,Amer. J. Pathol. 138:1379, 1991; Godfrey et al., Proc. Natl. Acad. Sci.USA 88:5528, 1991). Other clinical symptoms include elevated leukocytecounts, hypergammaglobulinemia, and severe anemia (Lyon et al., Proc.Natl. Acad. Sci. USA 87:2433, 1990); the death of affected males usuallyoccurs by 3 weeks of age. The sf locus has been mapped to the extremeproximal region of the X chromosome, approximately 0.7 centimorgans fromthe locus for sparse-fur (spf) (Lyon et al., Proc. Natl. Acad. Sci. USA87:2433, 1990; Blair et al., Mamm. Genome 5:652, 1994), itself a pointmutation within the ornithine transcarbamylase gene (Otc) (Veres et al.,Science 237:415, 1987). The sf locus is also tightly linked to themurine Gata1, Tcfe3, and Wasp loci (Blair et al., Mamm. Genome 5:652,1994; Derry et al., Genomics 29:471, 1995). Similarities between scurfyand human Wiskott-Aldrich syndrome (WAS) have been noted (Lyon et al.,Proc. Natl. Acad. Sci. USA 87:2433, 1990), and the mouse Wasp gene hasbeen proposed as a candidate for scurry (Lyon et al., Proc. Natl. Acad.Sci. USA 87:2433, 1990; Derry et al., Genomics 29:471, 1995). Closerbiological examination reveals significant differences between WAS andscurfy, however, and the two loci have been demonstrated to benon-allelic (Jeffery & Brunkow, unpublished data). Thus, prior toapplicants' invention the identity of the scurfy gene remained to bedetermined.

The present invention discloses methods and compositions useful fordiagnosing scurfy-related diseases, as well as methods for identifyingcompounds which can modulate the immune system, and further providesother related advantages.

SUMMARY OF THE INVENTION

The present invention relates generally to the discovery of novel geneswhich, when mutated, results in a profound lymphoproliferative disorder.In particular, a mutant mouse, designated ‘Scurfy’, was used to identifythe gene responsible for this disorder through backcross analysis,physical mapping and large-scale DNA sequencing. Analysis of thesequence of this gene indicated that it belongs to a family of relatedgenes, all containing a winged-helix DNA binding domain.

Thus, within one aspect of the invention isolated nucleic acid moleculesare provided which encode Fkh^(sf) or Fkh^(sf), including mutant formsthereof. Within certain embodiments, Fkh^(sf) of any type may be from awarm-blooded animal, such as a mouse or human. Within furtherembodiments, isolated nucleic acid molecules are provided wherein thenucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule that encodes an amino acid sequence comprising SEQID Nos 2, or, 4,(b) a nucleic acid molecule that hybridizes understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID Nos: 1, or, 3, or its complement, and (c) a nucleicacid molecule that encodes a functional fragment of the polypeptideencoded by either (a) or (b). Preferably, the nucleic acid molecule isnot JM2. Within related aspects, vectors (including expression vectors),and recombinant host cells are also provided, as well as proteins whichare encoded by the above-noted nucleic acid molecules. Further, fusionproteins are also provided which combine at least a portion of theabove-described nucleic acid molecules with the coding region of anotherprotein. Also provided are oligonucleotide fragments (including probesand primers) which are based upon the above sequence. Such fragments areat least 8, 10, 12, 15, 20, or 25 nucleotides in length, and may extendup to 100, 200, 500, 1000, 1500, or, 2000 nucleotides in length.

Within other aspects methods of using the above noted expression vectorfor producing a Fkh^(sf) protein (of any type) are provided, comprisingthe general steps of (a) culturing recombinant host cells that comprisethe expression vector and that produce Fkh^(sf) protein, and (b)isolating protein from the cultured recombinant host cells.

Also provided are antibodies and antibody fragments that specificallybind to Fkh^(sf) proteins. Representative examples of such antibodiesinclude both polyclonal and monoclonal antibodies (whether obtained froma murine hybridoma, or derived into human form). Representative examplesof antibody fragments include F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv, andminimal recognition units or complementarity determining regions.

Within yet other aspects, methods are provided for detecting thepresence of a Fkh^(sf) nucleic acid sequence in a biological sample froma subject, comprising the steps of (a) contacting a Fkh^(sf) specificnucleic acid probe under hybridizing conditions with either (i) testnucleic acid molecules isolated from said biological sample, or (ii)nucleic acid molecules synthesized from RNA molecules, wherein saidprobe recognizes at least a portion of nucleotide sequence of claim 1,and (b) detecting the formation of hybrids of said nucleic acid probeand (i) or (ii).

Within another related embodiment, methods are provided for detectingthe presence of an Fkh^(sf), or a mutant form thereof, in a biologicalsample, comprising the steps of: (a) contacting a biological sample withan anti-Fkh^(sf) antibody or an antibody fragment, wherein saidcontacting is performed under conditions that allow the binding of saidantibody or antibody fragment to said biological sample, and (b)detecting any of said bound antibody or bound antibody fragment.

Within other aspects of the invention, methods are provided forintroducing Fkh^(sf) nucleic acid molecules to an animal, comprising thestep of administering a Fkh^(sf) nucleic acid molecule as describedherein to an animal (e.g., a human, monkey, dog, cat, rat, or, mouse.Within one embodiment, the nucleic acid molecule is contained within andexpressed by a viral vector (e.g., a vector generated at least in partfrom a retrovirus, adenovirus, adeno-associated virus, herpes virus, or,alphavirus). Within another embodiment the nucleic acid molecule isexpressed by, or contained within a plasmid vector. Such vectors may beadministered either in vivo, or ex vivo (e.g., to hematopoietic cellssuch as T cells.

Within other embodiments, transgenic non-human animals are providedwherein the cells of the animal express a transgene that contains asequence encoding Fkh^(sf) protein.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a nucleotide sequence of mouse Fkh^(sf) cDNA (SeqeunceI.D. No. 1); translation is predicted to initiate at position 259 andterminate at position 1546.

FIG. 2 depicts the amino acid sequence of mouse Fkh^(sf) (Sequence I.D.No. 2).

FIG. 3 depicts a nucleotide sequence of 1735 bp corresponding to humanFKHsf cDNA (Sequence I.D. No. 3; including a 1293 bp coding region);translation is predicted to initiate at position 55 and terminate atposition 1348.

FIG. 4 depicts the sequence of a 431 amino acid human FKH^(sf) protein(Sequence I.D. No. 4).

FIG. 5 diagrammatically depicts a vector for generation of FKH^(sf)transgenic mice.

FIG. 6 is a photograph which demonstrates that the FKH^(sf) transgenecorrects the defect in scurfy animals.

FIG. 7 is a graph which shows that FKH^(sf) tg mice have reduced lymphnode cells, as compared to normal cells.

FIG. 8 is a graph which shows that FKH^(sf) transgenic mice respondpoorly to in vitro stimulation.

FIG. 9 is a comparison of FKH^(sf) and JM2 cDNAs.

FIG. 10 compares homology in various regions of human FKH^(sf) andmurine Fkh^(sf).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Prior to setting forth the Invention in detail, it may be helpful to anunderstanding thereof to set forth definitions of certain terms and tolist and to define the abbreviations that will be used hereinafter.

“Scurfy” refers to an inherited disease in mice which exhibit a severelymphoproliferative disorder (see, e.g., Lyon et al., Proc. Natl. Acad.Sci. USA 87:2433, 1990). The responsible gene (mutant forms of which areresponsible for the disease) is shown in Sequence I.D. Nos. 1 and 3.

“Molecule” should be understood to include proteins or peptides (e.g.,antibodies, recombinant binding partners, peptides with a desiredbinding affinity), nucleic acids (e.g., DNA, RNA, chimeric nucleic acidmolecules, and nucleic acid analogues such as PNA), and organic orinorganic compounds.

“Nucleic acid” or “nucleic acid molecule” refers to any ofdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides,fragments generated by the polymerase chain reaction (PCR), andfragments generated by any of ligation, scission, endonuclease action,and exonuclease action. Nucleic acids can be composed of monomers thatare naturally-occurring nucleotides (such as deoxyribonucleotides andribonucleotides), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have modifications insugar moieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleicacid” also includes so-called “peptide nucleic acids,” which comprisenaturally-occurring or modified nucleic acid bases attached to apolyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

“Isolated nucleic acid molecule” is a nucleic acid molecule that is notintegrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a gene that has been separated from the genomicDNA of a eukaryotic cell is an isolated DNA molecule. Another example ofan isolated nucleic acid molecule is a chemically-synthesized nucleicacid molecule that is not integrated in the genome of an organism.

“Promoter” is a nucleotide sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter.

“Vector” refers to an assembly which is capable of directing theexpression of desired protein. The vector must include transcriptionalpromoter elements which are operably linked to the genes of interest.The vector may be composed of either deoxyribonucleic acids (“DNA”),ribonucleic acids (“RNA”), or a combination of the two (e.g., a DNA-RNAchimeric). Optionally, the vector may include a polyadenylationsequence, one or more restriction sites, as well as one or moreselectable markers such as neomycin phosphotransferase or hygromycinphosphotransferase. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements such as an origin ofreplication, additional nucleic acid restriction sites, enhancers,sequences conferring inducibility of transcription, and selectablemarkers, may also be incorporated into the vectors described herein.

“Isolated” in the case of proteins or polypeptides, refers to moleculeswhich are present in the substantial absence of other biologicalmacromolecules, and appear nominally as a single band on SDS-PAGE gelwith coomassie blue staining. “Isolated” when referring to organicmolecules means that the compounds are greater than 90% pure utilizingmethods which are well known in the art (e.g., NMR, melting point).

“Cloning vector” refers to nucleic acid molecules, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites at whichforeign nucleotide sequences can be inserted in a determinable fashionwithout loss of an essential biological function of the vector, as wellas nucleotide sequences encoding a marker gene that is suitable for usein the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance or ampicillin resistance.

“Expression vector” refers to a nucleic acid molecule encoding a genethat is expressed in a host cell. Typically, gene expression is placedunder the control of a promoter, and optionally, under the control of atleast one regulatory element. Such a gene is said to be “operably linkedto” the promoter. Similarly, a regulatory element and a promoter areoperably linked if the regulatory element modulates the activity of thepromoter.

“Recombinant host” refers to any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Fkh^(sf)” or a “Fkh^(sf)anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the gene, or (b)capable of forming a stable duplex with a portion of an mRNA transcript.Similarly, an “anti-sense oligonucleotide specific for “Fkh^(sf)” or a“Fkh^(sf) anti-sense oligonucleotide” is an oligonucleotide having asequence (a) capable of forming a stable triplex with a portion of theFkh^(sf) gene, or (b) capable of forming a stable duplex with a portionof an mRNA transcript of the Fkh^(sf) gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

Abbreviations: YAC, yeast artificial chromosome; PCR, polymerase chainreaction; RT-PCR, PCR process in which RNA is first transcribed into DNAat the first step using reverse transcriptase (RT); cDNA, any DNA madeby copying an RNA sequence into DNA form. As utilized herein “Fkh^(sf)”refers to the gene product of the Fkh^(sf) gene (irrespective of whetherthe gene is obtained from humans, mammals, or any other warm-bloodedanimal). When capitalized “FKH^(sf)” the gene product (and gene) shouldbe understood to be derived from humans.

As noted above, the present invention relates generally topharmaceutical products and methods and, more specifically, to methodsand compositions useful for diagnosing scurfy-related diseases, as wellas methods for identifying compounds which can modulate the immunesystem.

Thus, as discussed in more detail below this discovery has led to thedevelopment of assays which may be utilized to select molecules whichcan act as agonists, or alternatively, antagonists of the immune system.Furthermore, such assays may be utilized to identify other genes andgene products which are likewise active in modulating the immune system.

Scurfy

Briefly, the present inventions are based upon the unexpected discoverythat a mutation in the gene which encodes Fkh^(sf) results in rarecondition (scurfy) characterized by a progressive lymphocyticinfiltration of the lymph nodes, spleen, liver and skin resulting ingross morphological symptoms which include splenomegaly, hepatomegaly,greatly enlarged lymph nodes, runting, exfoliative dermatitis, andthickened malformed ears (Godfrey et al., Amer. J. Pathol. 138:1379,1991; Godfrey et al., Proc. Natl. Acad. Sci. USA 88:5528, 1991). Thisnew member of the winged-helix family represents a novel component ofthe immune system.

Methods which were utilized to discover the gene responsible for scurfyare provided below in Example 1. Methods for cloning the generesponsible for murine scurfy, as well as the human ortholog, areprovided below in Examples 2 and 3. Methods for confirmation of geneidentity and correlation with gene function, as determined usingtransgenic mice, are also provided in the Examples.

Also provided by the present invention are methods for determining thepresence of Fkh^(sf) genes and gene products. Within one embodiment,such methods comprise the general steps of (a) contacting a Fkh^(sf)specific nucleic acid probe under hybridizing conditions with either (i)test nucleic acid molecules isolated from the biological sample, or (ii)nucleic acid molecules synthesized from RNA molecules, wherein the proberecognizes at least a portion of an Fkh^(sf) nucleotide sequence, and(b) detecting the formation of hybrids of said nucleic acid probe and(i) or (ii). A variety of methods may be utilized in order to amplify aselected sequence, including, for example, RNA amplification (seeLizardi et al., Bio/Technology 6:1197–1202, 1988; Kramer et al., Nature339:401–402, 1989; Lomeli et al., Clinical Chem. 35(9):1826–1831, 1989;U.S. Pat. No. 4,786,600), and nucleic acid amplification utilizingPolymerase Chain Reaction (“PCR”) (see U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159), reverse-transcriptase-PCR and CPT (see U.S.Pat. Nos. 4,876,187, and 5,011,769).

Alternatively, antibodies may be utilized to detect the presence ofFkh^(sf) gene products. More specifically, within one embodiment methodsare provided for detecting the presence of an Fkh^(sf) peptide, or amutant form thereof, in a biological sample, comprising the steps of (a)contacting a biological sample with an anti-Fkh^(sf) antibody or anantibody fragment, wherein said contacting is performed under conditionsthat allow the binding of said antibody or antibody fragment to thebiological sample, and (b) detecting any of the bound antibody or boundantibody fragment.

Such methods may be accomplished in a wide variety of assay formatsincluding, for example, Countercurrent Immuno-Electrophoresis (CIEP),Radioimmunoassays, Radioimmunoprecipitations, Enzyme-LinkedImmuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition orCompetition assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110and 4,486,530; see also Antibodies: A Laboratory Manual, supra).

Nucleic Acid Molecules, Proteins, And Methods of Producing Proteins

Although various FKH^(sf) or Fkh^(sf) proteins and nucleic acidmolecules (or portions thereof) have been provided herein, it should beunderstood that within the context of the present invention, referenceto one or more of these proteins should be understood to includeproteins of a substantially similar activity. As used herein, proteinsare deemed to be “substantially similar” if: (a) they are encoded by anucleotide sequence which is derived from the coding region of a genewhich encodes the protein (including, for example, portions of thesequence or allelic variations of the sequence); (b) the nucleotidesequence is capable of hybridization to nucleotide sequences of thepresent invention under moderate, high or very high stringency (seeSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, NY, 1989), or has at least 50%, 60%,70%, 75%, 80%, 90%, 95%, or greater homology to the sequences disclosedherein, or, (c) the DNA sequences are degenerate as a result of thegenetic code to the DNA sequences defined in (a) or (b). Further, thenucleic acid molecule disclosed herein includes both complementary andnon-complementary sequences, provided the sequences otherwise meet thecriteria set forth herein. Within the context of the present invention,high stringency means standard hybridization conditions (e.g., 5×SSPE,0.5% SDS at 65° C., or the equivalent). For purpose of hybridization,nucleic acid molecules which encode the amino-terminal domain, zincfinger domain, middle domain, or forkhead domain (see Example 10) may beutilized.

The structure of the proteins encoded by the nucleic acid moleculesdescribed herein may be predicted from the primary translation productsusing the hydrophobicity plot function of, for example, P/C Gene orIntelligenetics Suite (Intelligenetics, Mountain View, Calif.), oraccording to the methods described by Kyte and Doolittle (J. Mol. Biol.157:105–132, 1982).

Proteins of the present invention may be prepared in the form of acidicor basic salts, or in neutral form. In addition, individual amino acidresidues may be modified by oxidation or reduction. Furthermore, varioussubstitutions, deletions, or additions may be made to the amino acid ornucleic acid sequences, the net effect of which is to retain or furtherenhance or decrease the biological activity of the mutant or wild-typeprotein. Moreover, due to degeneracy in the genetic code, for example,there may be considerable variation in nucleotide sequences encoding thesame amino acid sequence.

Other derivatives of the proteins disclosed herein include conjugates ofthe proteins along with other proteins or polypeptides. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins which may be added to facilitate purification oridentification of proteins (see U.S. Pat. No. 4,851,341, see also, Hoppet al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteins(e.g., FKH or Fkh-luciferase or FKH or Fkh-GFP) may be constructed inorder to assist in the identification, expression, and analysis of theprotein.

Proteins of the present invention may be constructed using a widevariety of techniques described herein. Further, mutations may beintroduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes a derivative having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredgene having particular codons altered according to the substitution,deletion, or insertion required. Exemplary methods of making thealterations set forth above are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 1985,12–19) Smith et al. (Genetic Engineering: Principles and Methods, PlenumPress, 1981); and Sambrook et al. (supra). Deletion or truncationderivatives of proteins (e.g., a soluble extracellular portion) may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al. (MolecularCloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, 1989).

Mutations which are made in the nucleic acid molecules of the presentinvention preferably preserve the reading frame of the coding sequences.Furthermore, the mutations will preferably not create complementaryregions that could hybridize to produce secondary mRNA structures, suchas loops or hairpins, that would adversely affect translation of themRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed mutants screened for indicative biological activity.Alternatively, mutations may be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes aderivative having the desired amino acid insertion, substitution, ordeletion. Mutations may be introduced for purpose of preserving orincreasing activity of the protein, or, for decreasing or disabling theprotein (e.g., mutant Fkh).

Nucleic acid molecules which encode proteins of the present inventionmay also be constructed utilizing techniques of PCR mutagenesis,chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83:3402–3406,1986), by forced nucleotide misincorporation (e.g., Liao and Wise Gene88:107–111, 1990), or by use of randomly mutagenized oligonucleotides(Horwitz et al., Genome 3:112–117, 1989).

The present invention also provides for the manipulation and expressionof the above described genes by culturing host cells containing a vectorcapable of expressing the above-described genes. Such vectors or vectorconstructs include either synthetic or cDNA-derived nucleic acidmolecules encoding the desired protein, which are operably linked tosuitable transcriptional or translational regulatory elements. Suitableregulatory elements may be derived from a variety of sources, includingbacterial, fungal, viral, mammalian, insect, or plant genes. Selectionof appropriate regulatory elements is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of regulatory elements include: a transcriptional promoter andenhancer or RNA polymerase binding sequence, a transcriptionalterminator, and a ribosomal binding sequence, including a translationinitiation signal.

Nucleic acid molecules that encode any of the proteins described abovemay be readily expressed by a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,insect, or plant cells. Methods for transforming or transfecting suchcells to express foreign DNA are well known in the art (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl.Acad. Sci. USA 75:1929–1933, 1978; Murray et al., U.S. Pat. No.4,801,542; Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S.Pat. No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel etal., U.S. Pat. No. 4,766,075; and Sambrook et al. Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989;for plant cells see Czako and Marton, Plant Physiol. 104:1067–1071,1994; and Paszkowski et al., Biotech. 24:387–392, 1992).

Bacterial host cells suitable for carrying out the present inventioninclude E. coli, B. subtilis, Salmonella typhimurium, and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,as well as many other bacterial species well known to one of ordinaryskill in the art. Representative examples of bacterial host cellsinclude DHSα (Stratagene, LaJolla, Calif.).

Bacterial expression vectors preferably comprise a promoter whichfunctions in the host cell, one or more selectable phenotypic markers,and a bacterial origin of replication. Representative promoters includethe β-lactamase (penicillinase) and lactose promoter system (see Changet al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studieret al., Meth. Enzymol. 185:60–89, 1990), the lambda promoter (Elvin etal., Gene 87:123–126, 1990), the trp promoter (Nichols and Yanofsky,Meth. in Enzymology 101:155, 1983) and the tac promoter (Russell et al.,Gene 20:231, 1982). Representative selectable markers include variousantibiotic resistance markers such as the kanamycin or ampicillinresistance genes. Many plasmids suitable for transforming host cells arewell known in the art, including among others, pBR322 (see Bolivar etal., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119(see Messing, Meth. in Enzymology 101:20–77, 1983 and Vieira andMessing, Gene 19:259–268, 1982), and pNH8A, pNH16a, pNH18a, andBluescript M13 (Stratagene, La Jolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others, Saccharomyces pombe, Saccharomycescerevisiae, the genera Pichia or Kluyveromyces and various species ofthe genus Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349).Suitable expression vectors for yeast and fungi include, among others,YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3(Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl.Acad. Sci. USA 76:1035–1039, 1978), YEp13 (Broach et al., Gene8:121–133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104–108, 1978)and derivatives thereof.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073–12080, 1980;Alber and Kawasaki, J. Mol. Appl. Genet. 1:419–434, 1982) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum,N.Y., 1982; Ammerer, Meth. Enzymol. 101:192–201, 1983). Examples ofuseful promoters for fungi vectors include those derived fromAspergillus nidulans glycolytic genes, such as the adh3 promoter(McKnight et al., EMBO J. 4:2093–2099, 1985). The expression units mayalso include a transcriptional terminator. An example of a suitableterminator is the adh3 terminator (McKnight et al., ibid., 1985).

As with bacterial vectors, the yeast vectors will generally include aselectable marker, which may be one of any number of genes that exhibita dominant phenotype for which a phenotypic assay exists to enabletransformants to be selected. Preferred selectable markers are thosethat complement host cell auxotrophy, provide antibiotic resistance orenable a cell to utilize specific carbon sources, and include leu2(Broach et al., ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3(Struhl et al., ibid.). Another suitable selectable marker is the catgene, which confers chloramphenicol resistance on yeast cells.

Techniques for transforming fungi are well known in the literature, andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75:1929–1933, 1978), Yelton et al. (Proc.Natl. Acad. Sci. USA 81:1740–1747, 1984), and Russell (Nature301:167–169, 1983). The genotype of the host cell may contain a geneticdefect that is complemented by the selectable marker present on theexpression vector. Choice of a particular host and selectable marker iswell within the level of ordinary skill in the art.

Protocols for the transformation of yeast are also well known to thoseof ordinary skill in the art. For example, transformation may be readilyaccomplished either by preparation of spheroplasts of yeast with DNA(see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment withalkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163,1983). Transformation of fungi may also be carried out usingpolyethylene glycol as described by Cullen et al. (Bio/Technology 5:369,1987).

Viral vectors include those which comprise a promoter that directs theexpression of an isolated nucleic acid molecule that encodes a desiredprotein as described above. A wide variety of promoters may be utilizedwithin the context of the present invention, including for example,promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, adenoviralpromoter (Ohno et al., Science 265:781–784, 1994), neomycinphosphotransferase promoter/enhancer, late parvovirus promoter (Koeringet al., Hum. Gene Therap. 5:457–463, 1994), Herpes TK promoter, SV40promoter, metallothionein IIa gene enhancer/promoter, cytomegalovirusimmediate early promoter, and the cytomegalovirus immediate latepromoter. Within particularly preferred embodiments of the invention,the promoter is a tissue-specific promoter (see e.g., WO 91/02805; EP0,415,731; and WO 90/07936). Representative examples of suitable tissuespecific promoters include neural specific enolase promoter, plateletderived growth factor beta promoter, human alpha1-chimaerin promoter,synapsin I promoter and synapsin II promoter. In addition to theabove-noted promoters, other viral-specific promoters (e.g., retroviralpromoters (including those noted above, as well as others such as HIVpromoters), hepatitis, herpes (e.g., EBV), and bacterial, fungal orparasitic (e.g., malarial) -specific promoters may be utilized in orderto target a specific cell or tissue which is infected with a virus,bacteria, fungus or parasite.

Mammalian cells suitable for carrying out the present invention include,among others: PC12 (ATCC No. CRL1721), N1E-115 neuroblastoma,SK-N-BE(2)C neuroblastoma, SHSY5 adrenergic neuroblastoma, NS20Y andNG108-15 murine cholinergic cell lines, or rat F2 dorsal root ganglionline, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL6281; BHK 570 cell line (deposited with the American Type CultureCollection under accession number CRL 10314)), CHO (ATCC No. CCL 61),HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573; Graham et al., J. Gen.Virol. 36:59–72, 1977) and NS-1 cells. Other mammalian cell lines may beused within the present invention, including Rat Hep I (ATCC No. CRL1600), Rat Hep II (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), Humanlung (ATCC No. CCL 75.1), Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCCNo. HB 8065), Mouse liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL9.1), SP2/0-Ag14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777), Jurkat(ATCC No. Tib 152) and RINm 5AHT₂B (Orskov and Nielson, FEBS229(1):175–178, 1988).

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Viral promoters include the cytomegalovirusimmediate early promoter (Boshart et al., Cell 41:521–530, 1985),cytomegalovirus immediate late promoter, SV40 promoter (Subramani etal., Mol. Cell. Biol. 1:854–864, 1981), MMTV LTR, RSV LTR,metallothionein-1, adenovirus E1a. Cellular promoters include the mousemetallothionein-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), amouse V_(κ) promoter (Bergman et al., Proc. Natl. Acad. Sci. USA81:7041–7045, 1983; Grant et al., Nuc. Acids Res. 15:5496, 1987) and amouse V_(H) promoter (Loh et al., Cell 33:85–93, 1983). The choice ofpromoter will depend, at least in part, upon the level of expressiondesired or the recipient cell line to be transfected.

Such expression vectors may also contain a set of RNA splice siteslocated downstream from the promoter and upstream from the DNA sequenceencoding the peptide or protein of interest. Preferred RNA splice sitesmay be obtained from adenovirus and/or immunoglobulin genes. Alsocontained in the expression vectors is a polyadenylation signal locateddownstream of the coding sequence of interest. Suitable polyadenylationsignals include the early or late polyadenylation signals from SV40(Kaufman and Sharp, ibid.), the polyadenylation signal from theAdenovirus 5 E1B region and the human growth hormone gene terminator(DeNoto et al., Nuc. Acids Res. 9:3719–3730, 1981). The expressionvectors may include a noncoding viral leader sequence, such as theAdenovirus 2 tripartite leader, located between the promoter and the RNAsplice sites. Preferred vectors may also include enhancer sequences,such as the SV40 enhancer. Expression vectors may also include sequencesencoding the adenovirus VA RNAs. Suitable expression vectors can beobtained from commercial sources (e.g., Stratagene, La Jolla, Calif.).

Vector constructs comprising cloned DNA sequences can be introduced intocultured mammalian cells by, for example, calcium phosphate-mediatedtransfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson,Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841–845,1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY,1987). To identify cells that have stably integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Preferred selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. Other selectable markersinclude fluorescent proteins such as GFP (green fluorescent protein) orBFP (blue fluorescent protein). The selectable marker may be anamplifiable selectable marker. Preferred amplifiable selectable markersare the DHFR gene and the neomycin resistance gene. Selectable markersare reviewed by Thilly (Mammalian Cell Technology, ButterworthPublishers, Stoneham, Mass., which is incorporated herein by reference).

Mammalian cells containing a suitable vector are allowed to grow for aperiod of time, typically 1–2 days, to begin expressing the DNAsequence(s) of interest. Drug selection is then applied to select forgrowth of cells that are expressing the selectable marker in a stablefashion. For cells that have been transfected with an amplifiable,selectable marker the drug concentration may be increased in a stepwisemanner to select for increased copy number of the cloned sequences,thereby increasing expression levels. Cells expressing the introducedsequences are selected and screened for production of the protein ofinterest in the desired form or at the desired level. Cells that satisfythese criteria can then be cloned and scaled up for production. Cellsmay also be selected for transfection based on their expression of GFPby sorting for GFP-positive cells using a flow cytometer.

Protocols for the transfection of mammalian cells are well known tothose of ordinary skill in the art. Representative methods includecalcium phosphate mediated transfection, electroporation, lipofection,retroviral, adenoviral and protoplast fusion-mediated transfection (seeSambrook et al., supra). Naked vector constructs can also be taken up bymuscular cells or other suitable cells subsequent to injection into themuscle of a mammal (or other animals).

Numerous insect host cells known in the art can also be useful withinthe present invention, in light of the subject specification. Forexample, the use of baculoviruses as vectors for expressing heterologousDNA sequences in insect cells has been reviewed by Atkinson et al.(Pestic. Sci. 28:215–224, 1990).

Numerous plant host cells known in the art can also be useful within thepresent invention, in light of the subject specification. For example,the use of Agrobacterium rhizogenes as vectors for expressing genes inplant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore)11:47–58, 1987).

Within related aspects of the present invention, proteins of the presentinvention, may be expressed in a transgenic animal whose germ cells andsomatic cells contain a gene which encodes the desired protein and whichis operably linked to a promoter effective for the expression of thegene. Alternatively, in a similar manner transgenic animals may beprepared that lack the desired gene (e.g., “knockout” mice). Suchtransgenics may be prepared in a variety non-human animals, includingmice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al.,Nature 315:680–683, 1985, Palmiter et al., Science 222:809–814, 1983,Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438–4442, 1985, Palmiterand Brinster, Cell 41:343–345, 1985, and U.S. Pat. Nos. 5,175,383,5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384).Briefly, an expression vector, including a nucleic acid molecule to beexpressed together with appropriately positioned expression controlsequences, is introduced into pronuclei of fertilized eggs, for example,by microinjection. Integration of the injected DNA is detected by blotanalysis of DNA from tissue samples. It is preferred that the introducedDNA be incorporated into the germ line of the animal so that it ispassed on to the animal's progeny. Tissue-specific expression may beachieved through the use of a tissue-specific promoter, or through theuse of an inducible promoter, such as the metallothionein gene promoter(Palmiter et al., 1983, ibid), which allows regulated expression of thetransgene.

Animals which produce mutant forms of Fkh^(sf) other than the naturallyoccurring scurfy mutant (“sf”), or in genetic backgrounds different fromthe naturally occurring mutant, may be readily produced given thedisclosure provided herein.

Proteins can be isolated by, among other methods, culturing suitablehost and vector systems to produce the recombinant translation productsof the present invention. Supernatants from such cell lines, or proteininclusions or whole cells where the protein is not excreted into thesupernatant, can then be treated by a variety of purification proceduresin order to isolate the desired proteins. For example, the supernatantmay be first concentrated using commercially available proteinconcentration filters, such as an Amicon or Millipore Pelliconultrafiltration unit. Following concentration, the concentrate may beapplied to a suitable purification matrix such as, for example, ananti-protein antibody bound to a suitable support. Alternatively, anionor cation exchange resins may be employed in order to purify theprotein. As a further alternative, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps may be employed tofurther purify the protein. Other methods of isolating the proteins ofthe present invention are well known in the skill of the art.

A protein is deemed to be “isolated” within the context of the presentinvention if no other (undesired) protein is detected pursuant toSDS-PAGE analysis followed by Coomassie blue staining. Within otherembodiments, the desired protein can be isolated such that no other(undesired) protein is detected pursuant to SDS-PAGE analysis followedby silver staining.

ASSASYS FOR SELECTING MOLECULES WHICH MODULATE IMMUNE SYSTEM

As noted above, the present invention provides methods for selectingand/or isolating molecules which are capable of modulating the immunesystem. Representative examples of suitable assays include the yeast andmammalian 2-hybrid systems (e.g., Dang et al., Mol. Cell. Biol. 11:954,1991; Fearon et al., Proc. Natl. Acad. Sci. USA 89:7958, 1992), DNAbinding assays, antisense assays, traditional protein binding assays(e.g., utilizing ¹²⁵I or time-resolved fluorescence),immunoprecipitation coupled with gel electrophoresis and direct proteinsequencing, transcriptional analysis of Fkh^(sf) regulated genes,cytokine production and proliferation assays.

For example, within one embodiment proteins that directly interact withFkh^(sf) can be detected by an assay such as a yeast 2-hybrid bindingsystem (see, e.g., U.S. Pat. Nos. 5,283,173, 5,468,614, 5,610,015, and5,667,973). Briefly, in a two-hybrid system, a fusion of a DNA-bindingdomain-Fkh^(sf) protein (e.g., GAL4- Fkh^(sf) fusion) is constructed andtransfected into a cell containing a GAL4 binding site linked to aselectable marker gene. The whole Fkh^(sf) protein or subregions ofFkh^(sf) may be used. A library of cDNAs fused to the GAL4 activationdomain is also constructed and co-transfected. When the cDNA in thecDNA-GAL4 activation domain fusion encodes a protein that interacts withFkh^(sf), the selectable marker is expressed. Cells containing the cDNAare then grown, the construct isolated and characterized. Other assaysmay also be used to identify interacting proteins. Such assays includeELISA, Western blotting, co-immunoprecipitations, in vitrotranscription/translation analysis and the like.

Within another aspect of the present invention, methods are provided fordetermining whether a selected molecule is capable of modulating theimmune system, comprising the steps of (a) exposing a selected candidatemolecule to cells which express Fkh^(sf), or, mutant Fkh^(sf), and (b)determining whether the molecule modulates the activity of Fkh^(sf), andthereby determining whether said molecule can modulate the immunesystem. Cells for such tests may derive from (a) normal lymphocytes, (b)cell lines engineered to overexpress the FKH^(sf) (or Fkh^(sf)) protein(or mutant forms thereof) or (c) transgenic animals engineered toexpress said protein. Cells from such transgenic mice are characterized,in part, by a hyporesponsive state including diminished cell number anda decreased responsiveness to various stimuli (e.g., Example 8).

It should be noted that while the methods recited herein may refer tothe analysis of an individual test molecule, that the present inventionshould not be so limited. In particular, the selected molecule may becontained within a mixture of compounds. Hence, the recited methods mayfurther comprise the step of isolating the desired molecule.Furthermore, it should be understood that candidate molecules can beassessed for their ability to modulate the immune system by a number ofparameters, including for example, T-cell proliferation, cytokineproduction, and the like.

CANDIDATE MOLECULES

A wide variety of molecules may be assayed for their ability to modulatethe immune system. Representative examples which are discussed in moredetail below include organic molecules, proteins or peptides, andnucleic acid molecules.

1. Organic Molecules

Numerous organic molecules may be assayed for their ability to modulatethe immune system. For example, within one embodiment of the inventionsuitable organic molecules may be selected either from a chemicallibrary, wherein chemicals are assayed individually, or fromcombinatorial chemical libraries where multiple compounds are assayed atonce, then deconvoluted to determine and isolate the most activecompounds.

Representative examples of such combinatorial chemical libraries includethose described by Agrafiotis et al., “System and method ofautomatically generating chemical compounds with desired properties,”U.S. Pat. No. 5,463,564; Armstrong, R. W., “Synthesis of combinatorialarrays of organic compounds through the use of multiple componentcombinatorial array syntheses,” WO 95/02566; Baldwin, J. J. et al.,“Sulfonamide derivatives and their use,” WO 95/24186; Baldwin, J. J. etal., “Combinatorial dihydrobenzopyran library,” WO 95/30642; Brenner,S., “New kit for preparing combinatorial libraries,” WO 95/16918;Chenera, B. et al., “Preparation of library of resin-bound aromaticcarbocyclic compounds,” WO 95/16712; Ellman, J. A., “Solid phase andcombinatorial synthesis of benzodiazepine compounds on a solid support,”U.S. Pat. No. 5,288,514; Felder, E. et al., “Novel combinatorialcompound libraries,” WO 95/16209; Lerner, R. et al., “Encodedcombinatorial chemical libraries,” WO 93/20242; Pavia, M. R. et al., “Amethod for preparing and selecting pharmaceutically useful non-peptidecompounds from a structurally diverse universal library,” WO 95/04277;Summerton, J. E. and D. D. Weller, “Morpholino-subunit combinatoriallibrary and method,” U.S. Pat. No. 5,506,337; Holmes, C., “Methods forthe Solid Phase Synthesis of Thiazolidinones, Metathiazanones, andDerivatives thereof,” WO 96/00148; Phillips, G. B. and G. P. Wei,“Solid-phase Synthesis of Benzimidazoles,” Tet. Letters 37:4887–90,1996; Ruhland, B. et al., “Solid-supported Combinatorial Synthesis ofStructurally Diverse β-Lactams,” J. Amer. Chem. Soc. 111:253–4, 1996;Look, G. C. et al., “The Identification of Cyclooxygenase-1 Inhibitorsfrom 4-Thiazolidinone Combinatorial Libraries,” Bioorg and Med. Chem.Letters 6:707–12, 1996.

2. Proteins and Peptides

A wide range of proteins and peptides make likewise be utilized ascandidate molecules for modulating the immune system.

a. Combinatorial Peptide Libraries

Peptide molecules which modulate the immune system may be obtainedthrough the screening of combinatorial peptide libraries. Such librariesmay either be prepared by one of skill in the art (see e.g., U.S. Pat.Nos. 4,528,266 and 4,359,535, and Patent Cooperation Treaty PublicationNos. WO 92/15679, WO 92/15677, WO 90/07862, WO 90/02809, or purchasedfrom commercially available sources (e.g., New England Biolabs Ph.D.™Phage Display Peptide Library Kit).

b. Antibodies

Antibodies which modulate the immune system may readily be preparedgiven the disclosure provided herein. Within the context of the presentinvention, antibodies are understood to include monoclonal antibodies,polyclonal antibodies, anti-idiotypic antibodies, antibody fragments(e.g., Fab, and F(ab′)₂, F_(v) variable regions, or complementaritydetermining regions). As discussed above, antibodies are understood tobe specific against Fkh^(sf) if they bind with a K_(a) of greater thanor equal to 10⁷M, preferably greater than of equal to 10⁸M. The affinityof a monoclonal antibody or binding partner, as well as inhibition ofbinding can be readily determined by one of ordinary skill in the art(see Scatchard, Ann. N.Y. Acad. Sci. 51:660–672, 1949).

Briefly, polyclonal antibodies may be readily generated by one ofordinary skill in the art from a variety of warm-blooded animals such ashorses, cows, various fowl, rabbits, mice, or rats. Typically, Fkh^(sf),or a unique peptide thereof of 13–20 amino acids (preferably conjugatedto keyhole limpet hemocyanin by cross-linking with glutaraldehyde) isutilized to immunize the animal through intraperitoneal, intramuscular,intraocular, or subcutaneous injections, in conjunction with an adjuvantsuch as Freund's complete or incomplete adjuvant. Following severalbooster immunizations, samples of serum are collected and tested forreactivity to the protein or peptide. Particularly preferred polyclonalantisera will give a signal on one of these assays that is at leastthree times greater than background. Once the titer of the animal hasreached a plateau in terms of its reactivity to the protein, largerquantities of antisera may be readily obtained either by weeklybleedings, or by exsanguinating the animal.

Monoclonal antibodies may also be readily generated using conventionaltechniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and4,411,993 which are incorporated herein by reference; see alsoMonoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, andAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988, which are also incorporated herein byreference).

Other techniques may also be utilized to construct monoclonal antibodies(see William D. Huse et al., “Generation of a Large CombinationalLibrary of the Immunoglobulin Repertoire in Phage Lambda,” Science246:1275–1281, December 1989; see also L. Sastry et al., “Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library,” Proc. Natl Acad. Sci. USA 86:5728–5732,August 1989; see also Michelle Alting-Mees et al., “Monoclonal AntibodyExpression Libraries: A Rapid Alternative to Hybridomas,” Strategies inMolecular Biology 3:1–9, January 1990).

A wide variety of assays may be utilized to determine the presence ofantibodies which are reactive against the Fkh^(sf) (or the mutant formsof Fkh^(sf) described herein), including for example countercurrentimmuno-electrophoresis, radioimmunoassays, radioimmunoprecipitations,enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, westernblots, immunoprecipitation, Inhibition or Competition Assays, andsandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see alsoAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988).

Once suitable antibodies have been obtained, they may be isolated orpurified by many techniques well known to those of ordinary skill in theart (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), ColdSpring Harbor Laboratory Press, 1988). Suitable techniques includepeptide or protein affinity columns, HPLC or RP-HPLC, purification onprotein A or protein G columns, or any combination of these techniques.

Antibodies of the present invention may be utilized not only formodulating the immune system, but for diagnostic tests (e.g., todetermine the presence of an FKH^(sf) or Fkh^(sf) protein or peptide),for therapeutic purpose, or for purification of proteins.

c. Mutant Fkh^(sf)

As described herein and below in the Examples, altered versions ofFkh^(sf), may be utilized to inhibit the normal activity of Fkh^(sf),thereby modulating the immune system (see generally, nucleic acidmolecules and proteins above).

Further mutant or altered forms of FKH^(sf) or Fkh^(sf) may be utilizedfor a wide variety of in vitro assays (e.g., in order to examine theaffect of such proteins in various models), or, for the development ofantibodies.

3. Nucleic Acid Molecules

Within other aspects of the invention, nucleic acid molecules areprovided which are capable of modulating the immune system. For example,within one embodiment antisense oligonucleotide molecules are providedwhich specifically inhibit expression of FKH^(sf) or Fkh^(sf) nucleicacid sequences, or, of mutant FKH^(sf) or Fkh^(sf) (see generally,Hirashima et al. in Molecular Biology of RNA: New Perspectives (M.Inouye and B. S. Dudock, eds., 1987 Academic Press, San Diego, p. 401);Oligonucleotides: Antisense Inhibitors of Gene Expression (J. S. Cohen,ed., 1989 MacMillan Press, London); Stein and Cheng, Science261:1004–1012, 1993; WO 95/10607; U.S. Pat. No. 5,359,051; WO 92/06693;and EP-A2-612844). Briefly, such molecules are constructed such thatthey are complementary to, and able to form Watson-Crick base pairswith, a region of transcribed Fkh^(sf) mRNA sequence. The resultantdouble-stranded nucleic acid interferes with subsequent processing ofthe mRNA, thereby preventing protein synthesis.

Within other aspects of the invention, ribozymes are provided which arecapable of inhibiting FKH^(sf) or Fkh^(sf), or mutant forms FKH^(sf) orFkh^(sf). As used herein, “ribozymes” are intended to include RNAmolecules that contain anti-sense sequences for specific recognition,and an RNA-cleaving enzymatic activity. The catalytic strand cleaves aspecific site in a target RNA at greater than stoichiometricconcentration. A wide variety of ribozymes may be utilized within thecontext of the present invention, including for example, the hammerheadribozyme (for example, as described by Forster and Symons, Cell48:211–220, 1987; Haseloff and Gerlach, Nature 328:596–600, 1988; Walbotand Bruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature334:585, 1988); the hairpin ribozyme (for example, as described byHaseloff et al., U.S. Pat. No. 5,254,678, issued Oct. 19, 1993 andHempel et al., European Patent Publication No.0 360 257, published Mar.26, 1990); and Tetrahymena ribosomal RNA-based ribozymes (see Cech etal., U.S. Pat. No. 4,987,071). Ribozymes of the present inventiontypically consist of RNA, but may also be composed of DNA, nucleic acidanalogs (e.g., phosphorothioates), or chimerics thereof (e.g.,DNA/RNA/RNA).

4. Labels

FKH^(sf) or Fkh^(sf), (as well as mutant forms thereof), or, any of thecandidate molecules described above and below, may be labeled with avariety of compounds, including for example, fluorescent molecules,toxins, and radionuclides. Representative examples of fluorescentmolecules include fluorescein, Phycobili proteins, such asphycoerythrin, rhodamine, Texas red and luciferase. Representativeexamples of toxins include ricin, abrin diphtheria toxin, cholera toxin,gelonin, pokeweed antiviral protein, tritin, Shigella toxin, andPseudomonas exotoxin A. Representative examples of radionuclides includeCu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111,I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211,Pb-212 and Bi-212. In addition, the antibodies described above may alsobe labeled or conjugated to one partner of a ligand binding pair.Representative examples include avidin-biotin, and riboflavin-riboflavinbinding protein.

Methods for conjugating or labeling the molecules described herein withthe representative labels set forth above may be readily accomplished byone of ordinary skill in the art (see Trichothecene Antibody Conjugate,U.S. Pat. No. 4,744,981; Antibody Conjugate, U.S. Pat. No. 5,106,951;Fluorogenic Materials and Labeling Techniques, U.S. Pat. No. 4,018,884;Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Pat.No. 4,897,255; and Metal Radionuclide Chelating Compounds for ImprovedChelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Methods InEnzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B,Jakoby and Wilchek (eds.), Academic Press, New York, p. 30, 1974; seealso Wilchek and Bayer, “The Avidin-Biotin Complex in BioanalyticalApplications,” Anal. Biochem. 171:1–32, 1988).

PHARMACEUTICAL COMPOSITIONS

As noted above, the present invention also provides a variety ofpharmaceutical compositions, comprising one of the above-describedmolecules which modulates the immune system, along with apharmaceutically or physiologically acceptable carrier, excipients ordiluents. Generally, such carriers should be nontoxic to recipients atthe dosages and concentrations employed. Ordinarily, the preparation ofsuch compositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, the pharmaceutical composition (or,‘medicament’) is provided in sterile, pyrogen-free form.

In addition, the pharmaceutical compositions of the present inventionmay be prepared for administration by a variety of different routes. Inaddition, pharmaceutical compositions of the present invention may beplaced within containers, along with packaging material which providesinstructions regarding the use of such pharmaceutical compositions.Generally, such instructions will include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) which may be necessary to reconstitute thepharmaceutical composition.

METHODS OF T REATMENT

The present invention also provides methods for modulating the immunesystem. Through use of the molecules described herein which modulate theimmune system, a wide variety of conditions in warm blooded animals maybe readily treated or prevented. Examples of warm-blooded animals thatmay be treated include both vertebrates and mammals, including forexample humans, horses, cows, pigs, sheep, dogs, cats, rats and mice.Such methods may have therapeutic value in patients with altered immunesystems. This would include such patients as those undergoingchemotherapy of those with various immunodeficiency syndromes, as wellas patients with a T cell mediated autoimmune disease. Therapeutic valuemay also be recognized from utility as a vaccine adjuvant.

Therapeutic molecules, depending on the type of molecule, may beadministered via a variety of routes in a variety of formulations. Forexample, within one embodiment organic molecules may be delivered byoral or nasal routes, or by injection (e.g., intramuscularly,intravenously, and the like).

Within one aspect, methods are provided for modulating the immunesystem, comprising the step of introducing into lymphoid cells a vectorwhich directs the expression of a molecule which modulates the immunesystem, and administering the vector containing cells to a warm-bloodedanimal. Within other related embodiments, the vector may be directlyadministered to a desired target location (e.g., the bone marrow).

A wide variety of vectors may be utilized for such therapeutic purposes,including both viral and non-viral vectors. Representative examples ofsuitable viral vectors include herpes viral vectors (e.g., U.S. Pat. No.5,288,641), adenoviral vectors (e.g., WO 94/26914, WO 93/9191 WO99/20778; WO 99/20773; WO 99/20779; Kolls et al., PNAS 91(1):215–219,1994; Kass-Eisler et al., PNAS 90(24):11498–502, 1993; Guzman et al.,Circulation 88(6):2838–48, 1993; Guzman et al., Cir. Res.73(6):1202–1207, 1993; Zabner et al., Cell 75(2):207–216, 1993; Li etal., Hum Gene Ther. 4(4):403–409, 1993; Caillaud et al., Eur. J.Neurosci. 5(10:1287–1291, 1993; Vincent et al., Nat. Genet.5(2):130–134, 1993; Jaffe et al., Nat. Genet. 1(5):372–378, 1992; andLevrero et al., Gene 101(2):195–202, 1991), adeno-associated viralvectors (WO 95/13365; Flotte et al., PNAS 90(22):10613–10617, 1993),baculovirus vectors, parvovirus vectors (Koering et al., Hum. GeneTherap. 5:457–463, 1994), pox virus vectors (Panicali and Paoletti, PNAS79:4927–4931, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm.193(2):653–660, 1993), and retroviruses (e.g., EP 0,415,731; WO90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat.No. 5,219,740; WO 93/11230; WO 93/10218). Viral vectors may likewise beconstructed which contain a mixture of different elements (e.g.,promoters, envelope sequences and the like) from different viruses, ornon-viral sources. Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below.

Within other embodiments of the invention, nucleic acid molecules whichencode a molecule which modulates the immune system (e.g., a mutantFkh^(sf), or, an antisense or ribozyme molecule which cleaves Fkh^(sf))may be administered by a variety of alternative techniques, includingfor example administration of asialoosomucoid (ASOR) conjugated withpoly-L-lysine DNA complexes (Cristano et al., PNAS 92122–92126, 1993),DNA linked to killed adenovirus (Curiel et al., Hum. Gene Ther.3(2):147–154, 1992), cytofectin-mediated introduction (DMRIE-DOPE,Vical, Calif.), direct DNA injection (Acsadi et al., Nature 352:815–818,1991); DNA ligand (Wu et al., J. of Biol. Chem. 264:16985–16987, 1989);lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413–7417,1989); liposomes (Pickering et al., Circ. 89(1):13–21, 1994; and Wang etal., PNAS 84:7851–7855, 1987); microprojectile bombardment (Williams etal., PNAS 88:2726–2730, 1991); and direct delivery of nucleic acidswhich encode the protein itself either alone (Vile and Hart, Cancer Res.53: 3860–3864, 1993), or utilizing PEG-nucleic acid complexes.

Representative examples of molecules which may be expressed by thevectors of present invention include ribozymes and antisense molecules,each of which are discussed in more detail above.

As will be evident to one of skill in the art, the amount and frequencyof administration will depend, of course, on such factors as the natureand severity of the indication being treated, the desired response, thecondition of the patient, and so forth. Typically, the compositions maybe administered by a variety of techniques, as noted above.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 IDENTIFICATION OF THE GENE RESPONSIBILE FOR THESCURFY MUTANT

A. Cloning of a Scurfy Gene

The original scurfy mutation arose spontaneously in the partially inbredMR stock at Oak Ridge National Laboratory (ORNL) in 1949. Backcrossanalysis was used to fine map the peri-centromeric region of the Xchromosome containing the mouse Scurfy mutation. A physical map coveringthe same region was generated concurrently through the isolation ofoverlapping yeast and bacterial artificial chromosomes (YACs and BACs).Once the candidate region was narrowed down to ˜500 kilobase pairs (kb),large-scale DNA sequencing was performed on 4 overlapping BAC clones.All the transcription units in this 500 kb region were identifiedthrough a combination of sequence database searching and the applicationof computer exon prediction programs. Candidate genes were then screenedfor Scurfy-specific mutations by comparing the sequences of cDNAsobtained by the Reverse Transcription-Polymerase Chain Reaction (RT-PCR)procedure from normal and Scurfy-derived RNA samples. In one gene,referred to here as Fkh^(sf), a two base pair (bp) insertion was foundin the coding region of the Scurfy cDNA, relative to the normal cDNA.The insertion was confirmed by comparing the DNA sequences of PCRproducts derived from the genomic DNA of several mouse strains,including the Scurfy mutant. Again, the two bp insertion was found onlyin the Scurfy sample, establishing-this as the probable cause of theScurfy defect.

The mouse Fkh^(sf) gene is contained within the BAC clone 8C22, and hasbeen completely sequenced. It spans ˜14 kb and contains 11 coding exons.The locations of exon breaks were initially identified by computeranalysis of the genomic DNA sequence, using the GenScan exon predictionprogram; exon locations were then confirmed by direct comparison of cDNAsequences derived from normal mouse tissues to the genomic sequence.

The length of cDNA obtained is 2160 bp; the coding region spans 1287 bpof that, encoding a protein of 429 amino acids. FIG. 1 shows thenucleotide sequence of the mouse Fkh^(sf) cDNA; translation is predictedto initiate at position 259 and terminate at position 1546. FIG. 2 showsthe amino acid sequence of mouse Fkh^(sf).

b. Generation of Fkh^(sf) Transgenic Mice.

The identity of the Fkh^(sf) gene as the true cause of the Scurfyphenotype was confirmed in transgenic mice. Briefly, a 30 kb fragment ofthe normal genomic DNA, including the ˜7 kb coding region of theFkh^(sf) gene, as well as ˜20 kb of upstream flanking sequences and ˜4kb of downstream sequences (FIG. 5) was microinjected into normal mouseone-cell embryos. Five individual founder animals were generated, eachwith distinct integrations, and a male animal from each transgenic linewas crossed to a female sf carriers. Male offspring carrying both thetransgene (normal Fkh^(sf)) and sf mutation (mutant Fkh^(sf)) wereanalyzed.

Analysis consisted of examination of animals for runting, scaly skin,fur abnormalities and other hallmarks of the scurfy phenotype. Inaddition, lymphoid tissues (thymus, spleen and nodes) were harvested andtheir size and cell number examined and compared to both normal animalsas well as scurfy mice. For all five transgenic lines, male sf progenythat carried the transgene were normal in size and weight and appearedhealthy in all respects. Lymph node size in these transgenic mice wassimilar to (or smaller than) that of normal animals (FIG. 6) and therewas no sign of activated T cells. These parameters are extremelydifferent from sf mice and indicate that addition of the normal Fkh^(sf)gene can overcome the defect found in scurfy mice, thus confirming thatthe mutation in the Fkh^(sf) gene is the cause of Scurfy disease.

Example 2 GENERATION OF F KH ^(sf) cDNA

A complementary DNA (cDNA) encoding the complete mouse Fkh^(sf) proteinmay be obtained by a reverse-transcriptase polymerase chain reaction(RT-PCR) procedure. More specifically, first-strand cDNA is generated byoligo dT priming 5 ug of total RNA from a suitable source (eg., mousespleen) and extending with reverse transcriptase under standardconditions (eg., Gibco/BRL SuperScript kit). An aliquot of thefirst-strand cDNA is then subjected to 35 cycles of PCR (94° C. for 30sec, 63° C. for 30 sec, 72° C. for 2 min) in the presence of the forwardand reverse primers (Forward primer: GCAGATCTCC TGACTCTGCC TTC(SEQ IDNO: 5); Reverse primer: GCAGATCTGA CAAGCTGTGT CTG(SEQ ID NO: 6) (0.2 mMfinal concentration), 60 mM Tris-HCl, 15 mM ammonium sulfate, 1.5 mMmagnesium chloride, 0.2 mM each dNTP and 1 unit of Taq polymerase.

Example 3 GENERATION OF THE HUMAN ORTHOLOG TO MURINE FKH ^(sf)

A human FKH^(sf) cDNA encoding the complete FKH^(sf) protein may beobtained by essentially the same procedure as described in Example 2. Inparticular, starting with total spleen RNA, and utilizing the followingoligonucleotide primers (Forward primer: AGCCTGCCCT TGGACAAGGA C(SEQ IDNO: 7); Reverse primer: GCAAGACAGT GGAAACCTCA C(SEQ ID NO: 8)), and thesame PCR conditions outlined above, except with a 60° C. annealingtemperature.

FIG. 3 shows the nucleotide sequence of the 1869 bp cDNA obtained todate (including an 1293 bp coding region); translation is predicted toinitiate at position 189 and terminate at position 1482. FIG. 4 showsthe sequence of the 431 amino acid human FKH.sup.sf protein. Comparisonof the predicted coding region of the human gene to the mouse cDNAsequence reveals nearly identical exon structure and 86.1% amino acidsequence identity across the entire protein.

Example 4 MTHODS FOR DETECTING SCURFY M UTATIONS

As noted above, the Scurfy mutation was originally discovered bydirectly sequencing cDNAs derived by RT-PCR of sf and normal mouse RNAsamples, and confirmed by sequencing the same region from genomic DNA.The nature of the mutation (i.e., a 2 bp insertion) lends itself to anumber of different mutation detection assays. The first is based ondifferential hybridization of oligonucleotide probes. Such ahybridization-based assay could allow quantitative analysis ofallele-specific expression.

As an example, a 360 bp DNA fragment is amplified from 1^(st) strandcDNA using the following oligos:

DMO5985 (forward): CTACCCACTGCTGGCAAATG (ntd. 825–844 of FIG. 1) (SEQ IDNO: 9) DMO6724 (reverse): GAAGGAACTATTGCCATGGCTTC (ntd 1221–1199) (SEQID NO: 10)

The PCR products are run on an 1.8% agarose gel, transferred to nylonmembrane and probed with end-labeled oligonucleotides that arecomplementary to the region corresponding to the site of theScurfy-specific 2 bp insertion. Two separate hybridization reactions areperformed to detect the normal and Scurfy PCR products, using theoligonucleotides below (the site of the 2 bp insertion is shown inbold):

Normal: ATGCAGCAAGAGCTCTTGTCCATTGAGG (SEQ ID NO: DMO7439 11) Scurfy:GCAGCAAGAGCTCTTTTGTCCATTGAGG (SEQ ID NO: DMO6919 12)

The Scurfy mutation can also be detected by a cold Single-StrandConformation Polymorphism (cSSCP) assay. In this assay, the same PCRproducts described above are run on 20% acrylamide (TBE) gels afterstrand denaturation. The Scurfy insertion causes a shift in strandmobility, relative to the normal sequence, and the separate strands aredetected after staining with ethidium bromide.

Example 5 FKH ^(sf) GENE EXPRESSION

Semi-quantitative RT-PCR has been used to analyze the pattern of mouseand human Fkh^(sf) gene expression in a wide variety of tissues and celllines. Levels of expression are normalized to the ubiquitously expressedDAD-1 gene. In short, the Fkh^(sf) gene is expressed, albeit at very lowlevels, in nearly every tissue examined thus far, including thymus,spleen, sorted CD4+ and CD4−CD8− T-lymphocytes, as well as kidney,brain, and various mouse and human T-cell lines and human tumors.Absence of expression, however, was noted in freshly sorted mouseB-cells.

As expected, no differences in level of expression were observed innormal vs. Scurfy tissues in the RT-PCR assays.

Example 6 IN VITRO EXPRESSION OF FKH ^(sf)

Full-length mouse and human Fkh^(sf) cDNAs, as well as varioussub-regions of the cDNAs are cloned into vectors which allow expressionin mammalian cells (such as the human Jurkat T-cell line), E. coli oryeast. The E. coli or yeast systems can be used for production ofprotein for the purpose of raising Fkh^(sf)-specific antibodies (seebelow).

Example 7 GENERATION OF ANTI-FKH ^(SF) ANTIBODIES

Protein expressed from vectors described in example 6 are used toimmunize appropriate animals for the production of FKH^(sf) specificantibodies. Either full length or truncated proteins can be used forthis purpose. Protein can be obtained, for example, from bacteria suchas E. coli, insect cells or mammalian cells. Animal species can includemouse, rabbit, guinea pig, chicken or other. Rabbit antisera specificfor FKH^(sf) has been generated, as determined by biochemicalcharacterization (immunoprecipitation and western blotting).

Example 8 ASSAY FOR FUNCTION OF AN FKH^(SF) GENE

Since loss of function of the FKH^(sf) protein results in the phenotypeobserved in scurfy animals (wasting, hyperactive immune responsivenessand death), assays are described for assessing excessive expression ofthe FKH^(sf) protein. Transgenic animals (described in Example 1) areexamined for their state of immune competence, using several differentparameters. Animals are examined for the number of lymphoid cellspresent in lymph nodes and thymus (FIG. 7) as well as the responsivenessof T cells to in vitro stimulation (FIG. 8).

Scurfy mutant animals have roughly twice as many cells in their lymphnodes as normal animals, whereas mice which express excess levels of thenormal FKH^(sf) protein contain roughly one-third as many cells (FIG.7). Further, the number of thymocytes is normal (FIG. 7) as is theircell surface phenotype as assessed by flow cytometry using standardantisera (not shown), indicating that there is no developmental defectassociated with excess FKH^(sf) protein.

Normal, scurfy and transgenic animals are further examined for theirproliferative responses to T cell stimulation. CD4+ T cells are reactedwith antibodies to CD3 and CD28 and their proliferative responsemeasured using radioactive thymidine incorporation. Whereas only scurfycells divide in the absence of stimulation, normal cells respond wellfollowing stimulation. FKH^(sf) transgenic cells also respond tostimulation, however the response is significantly less than that ofnormal cells (FIG. 8). This indicates that CD4+ T cells that expressexcess FKH^(sf) have a diminished capacity to respond to stimuli.

Example 9 HUMAN FKH^(SF) cDNA SEQUENCE IS RELATED to JM2

A modified version of the human FKH^(sf) cDNA sequence exists in theGenBank public sequence database. This sequence is called JM2 (GenBankacc. # AJ005891), and is the result of the application of exonprediction programs to the genomic sequence containing the FKH^(sf) gene(Strom, T. M. et al., unpublished—see GenBank acc. # AJ005891). Incontrast, the structure of the FKH^(sf) cDNA was determinedexperimentally. The GAP program of the Genetics Computer Group (GCG;Madison, USA) Wisconsin sequence analysis package was used to comparethe two sequences, and the differences are illustrated in FIG. 9. The 5′ends of the two sequences differ in their location within the context ofthe genomic DNA sequence, the second coding exon of FKH^(sf) is omittedfrom JM2, and the last intron of the FKH^(sf) gene is unspliced in theJM2 sequence. These differences result in a JM2 protein with a shorteramino-terminal domain, relative to FKH^(sf), and a large insertionwithin the forkhead domain (see below) at the carboxy-terminus.

Example 10 The FKH^(sf) Protein is Conserved Across Species

The FKH^(sf) protein can be divided into sub-regions, based on sequencemotifs that may indicate functional domains. The two principal motifs inFKH^(sf) are the single zinc finger (ZNF) of the C₂H₂ class in themiddle portion of the protein, and the forkhead, or winged-helix domainat the extreme carboxy-terminus of the protein. For the purposes ofcharacterizing the degree of homology between FKH^(sf) and otherproteins, we have split the protein up into four regions:

Amino-terminal domain: residues 1–197 of FIG. 2 residues 1–198 of FIG. 4Zinc finger domain: residues 198–221 of FIG. 2 residues 199–222 of FIG.4 Middle domain: residues 222–336 of FIG. 2 residues 223–336 of FIG. 4Forkhead domain: residues 337–429 of FIG. 2 residues 337–431 of FIG. 4

Using the Multiple Sequence Alignment program from the DNAStar sequenceanalysis package, the Lipman-Pearson algorithm was employed to determinethe degree of similarity between the human FKH^(sf) and mouse Fkh^(sf)proteins across these four domains. The results are shown in FIG. 10.The similarity indices ranged from 82.8% to 96.4%, indicating that thisprotein is very highly conserved across species.

Example 11 IDENTIFICATION OF NOVEL FKH ^(Sf)-RELATED GENES

The unique features of the FKH^(sf) gene sequence may be used toidentify other novel genes (and proteins) which fall into the samesub-class of forkhead-containing molecules. The FKH^(sf) protein isunique in its having a single zinc finger domain amino-terminal to theforkhead domain as well as in the extreme carboxy-terminal position ofthe forkhead domain. A degenerate PCR approach may be taken to isolatenovel genes containing a zinc finger sequence upstream of a forkheaddomain. By way of example, the following degenerate primers weresynthesized (positions of degeneracy are indicated by parentheses, and“I” indicates the nucleoside inosine):

Forward primer: CA(TC)GGIGA(GA)TG(CT)AA(GA)TGG (SEQ ID NO: 13) Reverseprimer: (GA)AACCA(GA)TT(AG)TA(AGT)AT(CT)TC(GA)TT (SEQ ID NO: 14)

The forward primer corresponds to a region within the zinc fingersequence and the reverse primer corresponds to a region in the middle ofthe forkhead domain. These primers were used to amplify first-strandcDNA produced as in Example 2 from a variety of human tissues (includingliver, spleen, brain, lung, kidney, etc.). The following PCR conditionswere used: forward and reverse primers at 0.2 mM final concentration, 60mM Tris-HCl, 15 mM ammonium sulfate, 1.5 mM magnesium chloride, 0.2 mMeach dNTP and 1 unit of Taq polymerase, subjected to 35 cycles (94° C.for 30 sec, 50° C. for 30 sec, 72° C. for 2 min). PCR products werevisualized on a 1.8% agarose gel (run in 1× TAE) and sub-cloned into theTA cloning vector (Invitrogen, Carlsbad, Calif.); individual clones weresequenced and used for further characterization of full-length cDNAs.

Alternatively, the unique regions of the FKH^(sf) gene (i.e., the“Amino-terminal” and “Middle” domains) may be used to screen cDNAlibraries by hybridization. cDNA libraries, derived from a variety ofhuman and/or mouse tissues, and propagated in lambda phage vectors (eg.,lambda gt11) were plated on agarose, plaques were transferred to nylonmembranes and probed with fragments derived from the unique regions ofthe FKH^(sf) gene. Under high stringency conditions (eg., hybridizationin 5×SSPE, 5× Denhardt's solution, 0.5% SDS at 65° C., washed in0.1×SSPE, 0.1% SDS at 65C) only very closely related sequences areexpected to hybridize (i.e., 90–100% homologous). Under lowerstringency, such as hybridization and washing at 45°–55° C. in the samebuffer as above, genes that are related to FKH^(sf) (65–90% homologous)may be identified. Based on results obtained from searching publicdatabases with the unique sequences of FKH^(sf) any genes identifiedthrough low- to mid-stringency hybridization experiments are expected torepresent novel members of a “FKH^(sf) family”.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated nucleic acid molecule having at least 95% identity toresidues 259 to 1548 of SEQ ID NO:1 or the complement thereof, whereinCD4⁺T cells over-expressing said nucleic acid sequence exhibits areduction in CD3 and CD28 mediated T-cell proliferation.
 2. The nucleicacid molecule of claim 1, wherein the identity is at least 98%.
 3. Avector comprising the nucleic acid molecule of claim
 1. 4. An isolatedhost cell transformed with the vector of claim
 3. 5. An isolated nucleicacid molecule having at least 95% identity to residues 189 to 1484 ofSEQ ID NO:3 or the complement thereof, wherein CD4⁺T cellsover-expressing said nucleic acid sequence exhibits a reduction in CD3and CD28 mediated T-cell proliferation.
 6. The nucleic acid molecule ofclaim 5, wherein the identity is at least 98%.
 7. A vector comprisingthe nucleic acid molecule of claim
 5. 8. An isolated host celltransformed with the vector of claim
 7. 9. An isolated nucleic acidmolecule having at least 95% identity to SEQ ID NO:1 or the complementthereof, wherein CD4⁺T cells over-expressing said nucleic acid sequenceexhibits a reduction in CD3 and CD28 mediated T-cell proliferation. 10.The isolated nucleic acid molecule of claim 9, wherein the identity isat least 98%.
 11. A vector comprising the nucleic acid molecule of claim9.
 12. A host cell transformed with the vector of claim
 11. 13. Anisolated nucleic acid molecule having at least 95% identity to SEQ IDNO:3 or the complement thereof, wherein CD4⁺T cells over-expressing saidnucleic acid sequence exhibits a reduction in CD3 and CD28 mediatedT-cell proliferation.
 14. The isolated nucleic acid molecule of claim13, wherein the identity is at least 98%.
 15. A vector comprising thenucleic acid molecule of claim
 13. 16. An isolated host cell transformedwith the vector of claim 15.